The molecular cloning of the hemolysin structural gene (hly locus) from E1 Tor biotype strains of Vibrio cholerae 0-1 has been accomplished in Escherichia coli K-12. Following subcloning and Bal31 nuclease deletion analysis, the hly locus was found to be localized to a ca. 2.3 kilobase (kb) insert. This DNA fragment was used to probe chromosomal digests from a variety of V. cholerae strain including both Hly+ and Hly- E1 Tor biotypes, as well as Hly- classical strains. In all cases, a single hybridizing band was observed. Analysis from four base pair recognition restriction endonuclease digestion of the 2.3 kb insert from Hly+ strains compared to Hly- E1 Tor and Hly- classical strains has shown that a small region on the putative promoter side of the cloned fragment is specifically altered. The specific aims of this research proposal are to determine the nucleotide base sequence of V. cholerae hemolysin structural gene from Hly+ E1 Tor strains and to compare that sequence with those derived from the cloned hly locus of both non-hemolytic (Hly-) E1 Tor and classical biotype strains of V. cholerae 0-1. In addition, a study of protein secretion by V. cholerae through the isolation, characterization, and genetic mapping of temperature-sensitive secretion-defective mutants will be initiated. Should the nucleotide base sequence analysis of the hemolysin structural gene indicate that hemolysis is synthesized in precursor form with a leader peptide, I will further examine hemolysin secretion by site-directed mutational analysis. Several recent reports have suggested that V. cholerae expresses a "new toxin" that may be a virulence determinant. This "new toxin" has physical properties which are identical to those of the hemolysin. Since all prototype live oral vaccine strains so far described give rise to a mild diarrheal disease, and since purified hemolysin has been shown to have a variety of toxic activities, including enterotoxigenic activity, the expression of hemolysis may play a role in the pathogenesis of these prototype vaccine strains. It is hoped that studies will contribute to the development of a safe, effective live oral vaccine against cholera, as well as add to our basic understanding of the molecular genetics of V. cholerae hemolysin and protein export by this pathogen.